PROJECT SUMMARY The HIV-1 356 bp 5? untranslated leader contains multiple biological functions including elements required for initiating proviral DNA transcription, viral RNA reverse transcription, viral splicing, viral dimerization, and viral packaging. Reverse transcription initiation, in particular, is a major bottleneck to HIV-1 replication that has not been exploited for antiretroviral (ARV) development. Researchers at Stanford University, including a co- investigator of this proposal have published a cryo-EM structure of the reverse transcriptase (RT) initiation complex comprising HIV-1 RT, tRNA3Lys, and a 101-nucleotide viral RNA fragment encompassing 5?-leader positions 123 to 223. The structure shows that tRNA3Lys refolds and stacks onto the viral RNA primer binding site to form a double-stranded helical structure in the RT cleft. The bulkiness of the viral RNA-tRNA complex forces the RT enzyme into a suboptimally active conformation that must navigate the highly structured 5?-leader. Determining whether the 5?-leader region is under RT inhibitor selective drug pressure has implications for treating HIV-1 and HIV-2, and for refining our understanding of the genetic barriers to RT inhibitor resistance. However, there have been no published studies of paired sequences from patients before and after ARV therapy. We hypothesize that in patients receiving RT inhibitors, it may be necessary for HIV-1 to develop 5?-leader mutations to accommodate the effects of known nucleoside RT inhibitor (NRTI)- or nonnucleoside RT inhibitor (NNRTI)-resistance mutations. We have performed Sanger sequencing of 5?-leader positions 47 to 356 of paired virus samples from 11 patients before and after developing the cytosine analog resistance mutation M184V and/or the thymidine analog resistance mutation T215Y. We identified 22 mutations in 7 of 11 sequenced virus pairs at 14 nucleotides spanning positions 123 to 223. These mutations were not distributed randomly: seven occurred at positions 200 and 201, polymorphic nucleotides adjacent to the primer binding site and seven occurred in a polymorphic loop between nucleotides 207 to 216. Sequences of additional paired viruses before and after the development of key NRTI- and NNRTI-resistance mutations are needed to confirm an association between RT and 5?-leader mutations. We plan to perform Illumina next-generation sequencing (NGS) of the 5?-leader encompassing positions 47 to 356 for 105 paired samples including (i) 20 developing M184V/I; (ii) 15 developing T215Y/F; (iii) 20 developing K65R, L74V/I, K70Q/E/N/T, and Q151M; (iv) 20 developing an NNRTI-resistance mutation; and (v) 30 untreated control patients with paired viruses not developing RT inhibitor-resistance mutations. NGS will make it possible to identify covarying 5?-leader nucleotides at positions with electrophoretic mixtures by Sanger sequencing and to obtain high quality sequence data even when viral quasispecies display length heterogeneity. Should this study demonstrate that the 5?-leader is under selective RT inhibitor pressure, this would influence how RT inhibitors are developed and how their genetic barriers to drug resistance are defined.